Bio gel p 4

SCC (<1 kDa) was partially purified by various procedures that separated it from other molecules based on size and shape. The first step was usually ultrafiltration through filter units with a 10 kDa and then a 3 kDa cutoff, or just the latter (MilliporeSigma^TM Amicon^TM Ultra). Volumes of plasma or urine filtered were usually 2 ml, and half the volume of the filtrate was collected. For secreted SCC, volumes were either 2 or 15–30 ml, and 95% was filtered. Dialysis was with tubing that had a 0.1–0.5 kDa cutoff (Biotech CE dialysis tubing, Repligen). SCC samples were concentrated by freeze drying, with >90% recovery. Size exclusion chromatography (SEC) was carried out with 24 ml columns of Superdex 30 or 30 Increase (GE Healthcare; separation range 100–7000 Da) in a BioRad DuoFlow FPLC system, or in 100 ml gravity-flow columns of Biogel P4 (separation range 800–4000 Da; BioRad). For FPLC, 200 μl of sample was applied, and 0.5 ml fractions were collected. For the Biogel P4 columns, 1 ml of sample was applied, and 1 ml fractions were collected. SEC was carried out mostly with nanopure water and sometimes with 20 mM K phosphate, pH 7.0. Recoveries were >90%.

BLX‐1002 and BLX‐1237 were graciously donated by Bexel Pharmaceuticals, Inc. (Union City, CA). LY‐294002 was purchased from Calbiochem (La Jolla, CA). Wortmannin, acetylcholine chloride, Fura‐2/acetoxymethylester (Fura‐2/AM) and EGTA were from Sigma‐Aldrich (St. Louis, MO). GLP‐1 (7–36) was from PolyPeptide Laboratories (Torrance, CA). Collagenase A was from Roche Diagnostics (Mannheim, Germany) and Bio‐Gel P‐4 (fine, 65 ± 20 μm, wet) was from Bio‐Rad Laboratories (Hercules, CA). Rat insulin ELISA kits were from Mercodia (Uppsala, Sweden). RPMI‐1640 culture medium and FBS were from Life Technologies Invitrogen (Paisley, UK).

The dHfFG was further fractionated by repeating the gel permeation chromatography (GPC) on the Bio-gel P10 (medium, 2 × 180 cm, Bio-Rad Laboratories, Irvine, CA, USA), Bio-gel P6 (medium, 2 × 180 cm, Bio-Rad) and Bio-gel P4 (fine, 2 × 150 cm, Bio-Rad) columns. Briefly, dHfFG (~0.8 g) was dissolved in 5 mL of deionized water, subjected to the Bio-gel P10 column equilibrated well with 0.2 M NaCl and then eluted with the same solution. The flow rate was approximately 12 mL/h, and 100 fractions (2 mL/tube) were collected. The absorbance of each fraction was measured by the sulfuric acid-phenol method at 482 nm. The fractions containing the uniform distribution (detected by HPGPC) were combined and then desalted by a Sephadex G-10 (1.5 × 100 cm) column. Finally, three homogeneous fragments from dHfFG were freeze-dried to give Fr-1, Fr-2 and Fr-3.

Enzyme preparations were fractionated using a modified scheme described elsewhere [27 (link)]. In brief, proteins contained in a crude enzyme sample were preliminary precipitated with ammonium sulfate (80% saturation at 25°C) followed by a desalting procedure on a Bio-Gel P-4 (Bio-Rad Laboratories, Hercules, CA, USA) with the use of 0.02 M bis-Tris/HCl buffer, pH 6.8. Enzyme fractionation was carried out by anion-exchange chromatography on a Source 15Q HR 16/5 column (Pharmacia, Uppsala, Sweden). A sample containing 10 mg of protein was applied on the column equilibrated with 0.02 M bis-Tris/HCl buffer, pH 6.8. The bound protein was eluted with a gradient of 0 to 0.75 M NaCl at a flow rate of 1 mL/min (60 mL total volume). Protein concentration in collected fractions was determined by the modified Lowry method [28 (link)], using bovine serum albumin as the standard. Protein content in fractions containing the target heterologous enzymes (AnBGL or TrLPMO) was used to assay their content in the initial crude enzyme samples.